JP2018519853A5

JP2018519853A5 -

Info

Publication number: JP2018519853A5
Application number: JP2018513926A
Authority: JP
Filing date: 2016-05-27
Publication date: 2019-06-27

Claims

A method of altering the content of one or more cannabinoids in an asa plant, comprising downregulating the activity of tetrahydrocannabinolic acid (THCA) synthetase in the assassin plant.

Varying the content of cannabinoids reduces the THC content in the assemblage plant relative to delta-9-tetrahydrocannabinol (THC) content or cannabidiol (CBD) content in which the activity of TCHA synthetase is not down-regulated thereby, or that you increase the CBD content, the method of claim 1.

The down-regulation involves introducing into the plant an antisense RNA or siRNA selected to be effective for suppressing the expression of THCA synthetase, the selected DNA acting on the heterologous promoter The method of claim 1, wherein the method is operably linked.

(I) siRNA is
(A) a polynucleotide sequence having at least 85% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 61, 63, 64, 69, 71, 72, 68 and their complements;
(B) a polynucleotide sequence comprising at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 61, 63, 64, 69, 71, 72, 68 and their complements; or
(C) the THCA synthase, a polynucleotide sequence comprising a nucleotide, or the complement thereof to at least 21 consecutive, the sequence THCA synthase is selected from the group consisting of SEQ ID NO: 1-47, at least having 85% sequence identity, and is thus encoded by the polynucleotide sequence; or
(Ii) an antisense RNA,
(A) a polynucleotide sequence comprising SEQ ID NO: 81 or a fragment thereof capable of reducing the expression of THCA synthetase; or
(B) encoded by a polynucleotide sequence at least 85% identical to at least 200 contiguous nucleotides of SEQ ID NO: 81; and
4. The method of claim 3, wherein transcription of the selected DNA suppresses expression of THCA synthetase .

Down-regulating the activity of THCA synthetase
(A) Asa plant or cells thereof (i) at least one RNA-inducible endonuclease comprising at least one nuclear localization signal, or at least one RNA-inducing endonuclease comprising at least one nuclear localization signal nucleic acid encoding, and (ii) at least one guide RNA or introducing DN a encoding at least one guide RNA,; and,
(B) Assassins or their cells are cultured such that each guide RNA directs the RNA inducible endonuclease to the target site in the chromosomal sequence, where the RNA inducible endonuclease cleaves a double strand at the target site The introduced double-strand break is repaired by a DNA repair process so as to alter the chromosomal sequence, where the target site is located in the THCA synthetase gene, the chromosomal alteration is the THCA synthetase gene transcription and And / or preventing or inhibiting translation,
The method of claim 1, comprising:

The method according to claim 5, wherein in (a), (iii) at least one donor polynucleotide is further introduced into the plant or cells thereof.

(I) The RNA-inducible endonuclease is from a clustered equally-spaced short batch repeat (Prisins) / Pensor related (Cas) system ; or
(Ii) The method according to claim 5 or 6, wherein down-regulating the activity of THCA synthetase does not insert foreign genetic material, thereby producing a non-naturally occurring plant or cell thereof .

Down-regulating the activity of THCA synthetase
(A) identifying the locus of at least one THCA synthetase in the DNA sequence of the assassin plant or its cells;
(B) identifying at least one custom endonuclease recognition sequence at a locus of at least one THCA synthetase;
(C) At least a first custom endonuclease is introduced into the assassin plant or cells thereof, wherein the assassin plant or cells thereof recognize the custom endonuclease site within or near the locus of THCA synthetase Containing and transient or stable expression of custom endonucleases;
(D) assaying the Asa plant or cells thereof for custom endonuclease-mediated modification in the DNA constituting the THCA synthetase locus or in the DNA adjacent thereto;
(E) identifying an Asa plant, its cells or their progeny as containing a modification at the THCA synthetase locus;
The method of claim 1, comprising:

A transgenic asa plant or a seed thereof produced by the method according to any of claims 1-8 .

Reduction of the delta-9-tetrahydrocannabinol (THC) content, an increase of cannabidiol (CBD) content, or have reduced and increase of the CBD content of THC content, tetrahydrocannabinol acid (THCA) decreased expression of synthase Hemp plants or seeds thereof, cells thereof or parts thereof .

A selected DNA encoding an antisense RNA or siRNA effective to inhibit expression of THCA synthetase, wherein the selected DNA is operably linked to a heterologous promoter, wherein the asa plant is selected compared to similar genotype of hemp plants not containing been DNA, reduction of THC content, increased CBD content, or a decrease and increase in CBD content of THC content, hemp plant or its claim 10, wherein Seed, its cells or parts thereof .

  An assembly plant product derived from the assembly plant according to claim 10, comprising:
  (A) cannabidiol acid and cannabidiol at a concentration of about 18% to about 60% or about 7% to about 30% by total weight;
  (B) Delta-9-tetrahydrocannabinol and tetrahydrocannabonic acid at a concentration of about 0% to about 3% by total weight; or
  (C) Asa oil, asa tincture, dried asa flower and / or dried asa leaf.